一般社団法人
日本細胞生物学会Japan Society for Cell Biology

【PFA+Triton】:◎【PFA条件】3%PFA/PBS　37℃10min【Triton条件】0.1%　Triton-X100/PBS

【PFA+Triton】:×,【IS】:○？【PFA条件】3%PFA/PBS　37℃10min【Triton条件】0.1%　Triton-X100/PBS

【PFA+Triton】:×,【IS】:○？【PFA条件】3%PFA/PBS　37℃10min【Triton条件】0.1%　Triton-X100/PBS

【PFA+Triton】:×,【IS】:○？,【TCA】:○【PFA条件】3%PFA/PBS　37℃10min【TCA条件】TCA 10%/H2O on ice 15min【Triton条件】0.1%　Triton-X100/PBS

IFはTCA固定
TCA fix is best for IF.

【PFA+Triton】:○,【PFA+Saponin】:○【PFA条件】3%PFA/PBS　37℃10min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

WB:バンドが二本
Two bands appear by WB.

【PFA+Triton】:○,【PFA+Saponin】:○【PFA条件】3%PFA/PBS　37℃10min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

WB:バンドが一本、お勧め
A unique band appear by WB. Recommended Ab.

【PFA+Triton】:○,【PFA+Saponin】:○【PFA条件】3%PFA/PBS　37℃10min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

【PFA+Triton】:×,【PFA+Saponin】:×,【MeOH 】:×,【PFA+70%EtOH 】:×,【PFA_digitonin】:△(Can Get B)【PFA条件】3%PFA/PBS　37℃10min【MeOH条件】MeOH 100% -20℃ 5min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS【digitonin条件】50ug/ml digitonin /PBS

3%PFA (in PBS), methanol

WB:Blocking/3%BSA-TBS, 1stAb/3%BSA-TBS, 2ndAb/3% skin milk-TBSが良い。

WBはcan get signal (TOYOBO)使用
Use Cangetsignal (TOYOBO) for WB

TCAのみOK
Visualized only by TCA fix.

弱すぎる
Too weak staining.

蛍光染色不可
Not suitable for IF.

蛍光染色不可
Not suitable for IF.

蛍光染色不可
Not suitable for IF.

【PFA+Triton】:◎,【MeOH】:△,【PFA_70%EtOH】:○【PFA条件】3%PFA/PBS　37℃10min【MeOH条件】MeOH 100% -20℃ 5min【Triton条件】0.1%　Triton-X100/PBS

MDCKII OK.

【4%PFA+0.2%Saponin】:○

【PFA+Triton】:×,【PFA+Saponin】:×,【IS】:×,【MeOH】:×【PFA条件】3%PFA/PBS　37℃10min【MeOH条件】MeOH 100% -20℃ 5min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

【PFA+Triton】:△,【PFA+Saponin】:○,【TCA】:○【PFA条件】3%PFA/PBS　37℃10min【TCA条件】TCA 10%/H2O on ice 15min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

WB：Rab11aのみ認識、IFで見えるendosomeのシグナルはKDで半分ぐらいしか消えない。
Only recognize Rab11a by WB. Only half of the endosome staining dissapears in Rab11a KD cells.

【PFA+Triton】:×,【PFA+Saponin】:×,【IS】:×,【MeOH】:×,【TCA】:○、◎(+CanGetSignal (from TOYOBO Inc.))、BlockingはO/N【PFA条件】3%PFA/PBS　37℃10min【MeOH条件】MeOH 100% -20℃ 5min【TCA条件】TCA 10%/H2O on ice 15min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

WB: Rab11a, b両方認識する
Recognize both Rab11a and Rab11b by WB.

【PFA+Triton】:○,【PFA+Saponin】:○,【IS】:×,【MeOH】:○【PFA条件】3%PFA/PBS　37℃10min【MeOH条件】MeOH 100% -20℃ 5min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

PFAでもいいが、saponinがbetter
Saponin permeabilization is better.

【PFA+Triton】:×,【PFA+Saponin】:×,【IS】:×,【MeOH】:×【PFA条件】3%PFA/PBS　37℃10min【MeOH条件】MeOH 100% -20℃ 5min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

endosome likeのsignalが見えるが、siRab4で消えない。
Recognize endosome-like signals which cannot disappear by siRab4.

【PFA+Triton】:×,【PFA+Saponin】:×,【IS】:×,【MeOH】:×【PFA条件】3%PFA/PBS　37℃10min【MeOH条件】MeOH 100% -20℃ 5min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

TCA おそらく26C4ど同クローン

4%PFA, TCA

TCA

TCA 染色は弱い
Weak staining

分裂溝はTCAのみだが弱い
Cleavage furrows can be weakly stained only by TCA fix.

分裂溝の染色はTCAのみ
Cleavage furrows can be stained only by TCA fix.

4%PFA (in PBS)

Rab27A,B共に認識
recognize both Rab27a and Rab27b

4%PFA (in PBS)

TCA

PFA+Triton or MeOH or TCA

4%PFA (in PBS)

4%PFA (in PBS)

Rab11A,B共に認識
recognize both Rab11a and Rab11b

TCA (Can Get Signal Immunostain 使用）
http://www.toyobo.co.jp/seihin/xr/lifescience/tech/upload/upld96/technical/canget96tr01.pdf
http://www.toyobo.co.jp/seihin/xr/lifescience/tech/upload/upld95/technical/canget95tr02.pdf

3%PFA (in PBS), methanol

Rab8a,b共に認識
Recognize both Rab8a and Rab8b

4%PFA (in PBS)

Rab11A,B共に認識
Recognize both Rab11a and Rab11b

【PFA+Triton】:×,【PFA+Saponin】:×,【IS】:×,【MeOH】:×【PFA条件】3%PFA/PBS　37℃10min【MeOH条件】MeOH 100% -20℃ 5min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

Saponinがbetter 汚い
Saponin permeabilization is better.

TCAのみOK
Visualized only by TCA fix.

4%PFA (in PBS)

MEF, NIH3T3ではIF不可。Rab33Aは認識せず。
Not suitable for IF in MEFs and 3T3. Not recognize Rab33A.

【Formalin in PBS+0.05%Triton】：○,
【Formalin in Ringer+0.05%Triton】：○

ホルマリンは37％ホルマリン液（ナカライ）をPBSやRingerで１/10に希釈
Formalin solution is made by dilution of 37% formalin (Nacalai Inc.) by PBS or Ringer's solution

MeOHでmidobodyのみわかる程度で弱い
Only midbody can be weakly visualized by MeOH fix.

弱い（midbodyのみ）
Only midbody can be weakly visualized.

【PFA+Triton】:×,【PFA+Saponin】:×,【IS】:×,【MeOH】:×【PFA条件】3%PFA/PBS　37℃10min【MeOH条件】MeOH 100% -20℃ 5min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

【PFA+Triton】:○,【TCA】:◎【PFA条件】3%PFA/PBS　37℃10min【TCA条件】TCA 10%/H2O on ice 15min【Triton条件】0.1%　Triton-X100/PBS

【PFA+Triton】:×,【PFA+Saponin】:×,【IS】:×,【MeOH】:×【PFA条件】3%PFA/PBS　37℃10min【MeOH条件】MeOH 100% -20℃ 5min【Triton条件】0.1%　Triton-X100/PBS【saponin条件】0.05%　saponin /PBS

TCAのみOK
Visualized only by TCA fix.

4%PFA, 1%PFA, MeOH, TCA

WB:Blocking/3%BSA-TBS, 1stAb/3%BSA-TBS, 2ndAb/3% skin milk-TBSが良い。

1%PFA, TCA, MeOH

3%PFA (in PBS), methanol

PC12細胞の免疫蛍光染色（3%PFA固定、0.05%saponinで膜のpermeabilize）よく染まった
Good staining for IF of PC12 with 3%PFA, 0.05%saponin

3%PFA (in PBS)

3%PFA in PBS

0.05% saponin

TCAのみOK
Visualized only by TCA fix.

3%PFA (in PBS), methanol

3%PFA (in PBS), methanol